Immunological-based diagnostic assays have a wide application in the medical field. In particular, they are important tools as an aid in detecting and monitoring a variety of disease conditions. The effectiveness of these types of assays lies in part in the specificity of components within the immune system such as T-lymphocytes. Notwithstanding this specificity, immunological-based diagnostics are not necessarily always sensitive enough to detect low grade infections, the presence of a persistent low level infection, to detect infections in subjects with active or latent infectious disease states or in subjects exhibiting immunodeficiency or any form of immunosuppression. Desirable performance characteristics of a cell mediated immune response assay, in particular for detecting an antigen specific T-cell response, include adequate sensitivity, specificity, reliability and reproducibility and furthermore, should be simple and rapid to perform.
One established form of an immunological-based diagnostic assay involves the stimulation of T-cells or other cells of the immune system with antigens followed by the detection of immune effector molecules such as IFN-gamma or other cytokines produced in response to the stimulation with the antigen. The immune effector molecules are detected using well-known techniques such as enzyme immunoassays, multiplex bead analysis, ELISA, ELISpot and flow cytometry. The presence or increase in the level of immune effector molecules can also be determined based on the RNA level. Such assays are e.g. useful for detecting disease-specific immune responses, in particular pathogen specific immune responses. Respective assays are commercially available under the trademark QuantiFERON (Registered Trademark; Cellestis Limited) and can be e.g. used to diagnose a pathogen infection or to monitor cell-mediated immunity against a disease.
Other applications of respective cell-mediated immune response assays include the analysis or the monitoring of cellular immune responses to vaccines or immunotherapy, such as e.g. cancer immunotherapy.
There is a great demand for respective assays with enhanced sensitivity. Previously, methods for measuring cell-mediated immune responses were improved by incubating the sample such as a whole blood sample with the antigen in the presence of a simple sugar such as dextrose (see e.g. WO 2004/042396 A1). It was found that simple sugars such as dextrose and glucose increase the production of IFN-gamma by the immune cells and thereby improve the sensitivity of the assay. The use of simple sugars such as glucose and dextrose was believed to be essential to allow the cells to make use of that energy source and thus benefit from the addition of the sugar during incubation with the antigen. Significant increases in the INF-γ level were observed when the simple sugar was directly added to the sample in addition to the antigen. However, to simplify the performance of the method it is preferred to provide ready-to-use reagent compositions and to avoid manual handling steps. Therefore, it would be desirable to provide a single composition which includes the antigen and the simple sugar. Said composition could be provided in a sample collection tube, whereby the sample is directly contacted with the antigen and the simple sugar in the right concentration thereby avoiding handling errors. Here it was found though that the assay activity diminished over time when respective sample collection tubes comprising the antigen and the simple sugar were stored at room temperature or at elevated temperatures. This was found with certain antigens. Therefore, the shelf-life of these kit components is limited and after a certain storage time, the assay provides only low sensitivity or the assay activity is even completely lost. Therefore, respective kits/assay materials, wherein the antigen is conveniently provided together with a simple sugar in one composition, are not storage-stable and thus pose the risk that the assay sensitivity is reduced or even lost over time. This was not seen when the simple sugar was not included together with the antigen in the sample collection tube, but was added separately to the sample for incubation with the antigen.
The object of the present invention is to overcome at least one drawback of the prior art methods. In particular, it is the object of the present invention to provide sensitive and reliable methods for measuring cell-mediated immune responsiveness as well as kits and kit components which are storage-stable.